Chiral capillary electrophoresis as predictor for separation of drug enantiomers in continuous flow zone electrophoresis

Separation of the enantiomers of chlorpheniramine and methadone in acidic buffers containing carboxymethyl-betacyclodextrin (CMCD) as chiral selector was investigated by capillary zone electrophoresis. For a range of pH and CMCD concentrations, the mobility difference and resolution of the enantiomers were determined. Then, conditions known to provide well resolved enantiomers and optimized chiral separation were applied to chiral continuous flow electrophoresis. In that approach, a thin film of fluid flowing between two parallel plates is employed as carrier for electrophoresis. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. The number of pure enantiomeric fractions obtained by chiral continuous flow electrophoresis was found to be directly dependent on the enantiomeric mobility difference. For racemic chlorpheniramine separated in a betaine-acetic acid buffer at a total throughput of 5 mg/h, complete enantiomeric separation is shown to require a mobility difference of about 3 x 10(-9) m2/V s. Furthermore, compared to the previous investigations with hydroxypropyl-beta-cyclodextrin, CMCD was found to permit improved fractionation of methadone enantiomers. With a total racemic drug throughput of about 15 mg/h, continuous flow zone electrophoresis processing with CMCD as chiral selector is shown to have the potential of providing pure enantiomers on a mg/h scale. The results indicate that chiral capillary zone electrophoresis data can be employed as predictor for preparative scale chiral separations based upon continuous flow zone electrophoresis.     

毛细管区带电泳法拆分匹伐他汀钙对映体

建立了匹伐他汀钙对映体的毛细管区带电泳(CZE)拆分方法。分别考察了电泳电压,缓冲溶液种类、浓度及pH值,环糊精种类及浓度,添加剂种类及浓度等参数对实验结果的影响,从而确定了匹伐他汀钙对映体的最佳拆分条件: 电泳电压为18 kV;运行缓冲溶液为80 mmol/L的Tris-HCl缓冲体系,pH值为3.20,其中含有50 mmol/L HP-β-CD(羟丙基-β-环糊精)和5 mmol/L SDS(十二烷基磺酸钠);采用重力进样,进样高度17 cm,进样时间为2 s。在优化的实验条件下,匹伐他汀钙对映体得到了较好的分离,分离度可达2.17。实验结果表明该方法可用于匹伐他汀钙对映体的分离,具有快速、便捷、准确性好等优点。     

Chiral separation of highly negatively charged enantiomers by capillary electrophoresis

The separation of two highly negatively charged enantiomeric organic disulfates containing two chiral centers was investigated by capillary electrophoresis using cyclodextrin based chiral selectors added to the run buffer. The optimum separation for the enantiomers was achieved in less than 3 min at 25 degrees C with a run buffer of 10 mM glycine pH 2.4 and 5 mM QA-beta-CD, which is a positively charged quaternary ammonium beta-cyclodextrin derivative. The method resulted in baseline resolution, excellent linearity, and highly reproducible migration times allowing facile evaluation of the enantiomeric purity of the individual isomers. Detection limits for the enantiomeric pair were determined to be 0.3 ng/microl (S/N = 3). The nature of the selector-enantiomer interaction and a quantitative measurement of the apparent stability constants that governed chiral discrimination of the enantiomers with QA-beta-CD were also investigated by UV-Vis spectroscopy and electrospray ionization mass spectrometry.     

Chiral separations by capillary electromigration techniques in nonaqueous media. I. Enantioselective nonaqueous capillary electrophoresis

Enantiomer separations by CE employing nonaqueous conditions are reviewed. The general focus of this article is directed towards solvent effects on chiral recognition and the separation mechanism. After a general discussion of solvent effects on the individual processes involved in CE enantiomer separation, specifics for various selector classes are discussed together with a few applications of enantioselective nonaqueous CE.     

离子液体作为添加剂的毛细管电泳法拆分盐酸金刚乙胺

将新离子液体[EIMCH2CONHBu]BF4用于毛细管电泳法拆分手性药物,建立了以β-环糊精为手性选择剂拆分盐酸金刚乙胺对映体的毛细管电泳方法。分别考察了离子液体浓度,手性选择剂浓度,缓冲溶液种类、浓度及pH值,分离电压等参数对分离度的影响,从而确定了盐酸金刚乙胺对映体的最佳拆分条件:[EIMCH2CONHBu]BF4溶液体积分数3.2%,β-环糊精18 mmol/L,NaH2PO4 15 mmol/L,缓冲液pH 3.03,分离电压15 kV。在优化的实验条件下,盐酸金刚乙胺对映体得到基线分离,分离度可达1.51。实验结果表明[EIMCH2CONHBu]BF4能够增强β-环糊精的手性拆分能力,对手性拆分有协同作用。     

Rapid separation of basic drugs by nonaqueous capillary electrophoresis

Summary Nonaqueous capillary electrophoresis (NACE) has been used to achieve rapid separations of basic drugs. A high electric field was obtained by using short capillaries. Baseline separations of basic drugs, including amphetamines, tropane alkaloids and local anesthetics, were achieved in 1 min by selection of the appropriate organic solvent and electrolyte composition. Thus, high-throughput analyses can be performed. Peak efficiency up to 9154 theoretical plates s⁻¹ was achieved in a separation performed at 923V cm⁻¹. No discernible loss in resolution was observed when a conventional capillary (64.5cm) was replaced by a short (32.5 cm) capillary.     

Chiral separation of cetirizine by capillary electrophoresis

Chiral separation of cetirizine, a second-generation H(1)-antagonist, was studied by CD-mediated CE. Several parameters, including pH, CD type, buffer concentration, type of co-ion, applied voltage and temperature, were investigated. The best conditions for chiral separation were obtained using a 75 mM triethanolamine-phosphate buffer (pH 2.5) containing 0.4 mg/mL heptakis(2,3-diacetyl-6-sulfato)-beta-CD and 10% ACN. Online UV detection was performed at 214 nm, a voltage of 20 kV was applied and the capillary was temperature controlled at 25 degrees C by liquid cooling. Hydrodynamic injection was performed for 1 s. The method was validated for the quantification of levocetirizine in tablets and for enantiomeric purity testing of the drug substance. Selectivity, linearity, LOD and LOQ, precision and accuracy were evaluated for both methods. The amount of levocetirizine dihydrochloride in the commercially available tablets was quantified and was found to be within the specification limits of the claimed amount (5 mg). The amount of distomer in levocetirizine drug substance was found to be 0.87 +/- 0.09% w/w, which is in agreement with the certificate of analysis supplied by the company.     

Chiral separation of local anaesthetics with capillary electrophoresis

Summary A chiral capillary electrophoresis system for the highresolution separation of the enantiomers of the local anaesthetics mepivacaine, ropivacaine, bupivacaine and prilocaine is described. Triethanolamine was added to the background electrolyte to obtain a negative electroosmotic flow and hence higher resolutions. The interactions of the local anaesthetics and their chemical analogues with the chiral selector, dimethyl--cyclodextrin, were studied. From a model describing chiral capillary electrophoresis, the association equilibrium constants were determined by curve-fitting. The separation of mepivacaine, ropivacaine and bupivacaine was due to the different mobilities of the free analytes in solution, whereas the separation of a pair of enantiomers of a single analyte was due to differences between the association equilibrium constantsK ₁ andK ₂. Branching of the alkyl chain, which was situated close to the cavity in the inclusion complex, had strong effects on the chiral separation of the enantiomers.     

水相体系毛细管电泳与非水体系毛细管电泳分离手性药物的比较

将非水毛细管电泳(NACE)与传统的水相毛细管电泳从有机溶剂的添加、离子强度的改变以及pH的改变等方面作了一系列的对比性研究。在有机组分从10%增加到100%的过程中,被分离样品的迁移速率逐渐降低,其分离效率先呈升高的趋势,达到一个最大值后,在呈下降的趋势。这主要是由于缓冲介质粘度和介电常数的变化以及管壁Zeta电位的变化所造成的。在非水体系中,最佳分离pH要比在水相体系中高,其分离效率也是随着离子强度的增大而升高。     

Derivatized cyclodextrins for the separation of enantiomers in capillary electrophoresis

The use of cyclodextrin derivatives for the efficient separation of enantiomeric drugs is described. Hydroxypropylation, methylation or carboxymethylation of the cyclodextrin not only result in a better solubility of the cyclodextrin in aqueous solutions, but also favor, via additional hydrogen bonding, the stabilization of one of the cyclodextrin-analyte complexes. The influence of the background electrolyte on peak shape is also described here. Carboxymethylated cyclodextrin can be used in similar manner to uncharged cyclodextrins at low pH values (below 4). At pH values above 5, however, its charge also allows the separation of uncharged enantiomers as in a micellar-like system.     

Chiral separation by capillary electrophoresis with oligosaccharides.

Maltodextrins, i.e., mixtures of linear alpha-(1-4)-linked D-glucose polymers, were found to be effective as chiral electrolyte modifiers to perform direct, rapid separations by capillary electrophoresis of racemic mixtures of 2-arylpropionic acid non-steroidal anti-inflammatory compounds and coumarinic anticoagulant drugs, and also diastereomeric cephalosporin antibiotics. Enantioselectivity seemed to be dependent on an as yet unidentified combination of variables.     

Chiral separation of bupivacaine enantiomers by capillary electrophoresis partial-filling technique with human serum albumin as chiral selector

Capillary electrophoresis (CE) is a powerful technique for enantiomer separations due to its intrinsic high separation efficiencies, speed of analysis, low reagent consumption and small sample requirements. However, some chiral selectors present strong background UV absorption providing high detection limits. The present paper deals with the application of the partial-filling technique to the separation of bupivacaine enantiomers by capillary electrophoresis using human serum albumin (HSA) as chiral selector. In this procedure the cationic surfactant cetyltrimethylammonium bromide (CTAB) was used as a dinamic capillary coating in order to reduce the electro-osmotic flow and detect both bupivacaine enantiomers out of the chiral selector plug. Several experimental conditions such as CTAB concentration, pH, HSA concentration and plug length, background electrolyte concentration, temperature and voltage were studied. Under the selected conditions it is possible to detect the separated enantiomers out of the HSA plug in less than 4 min using 50 mM Tris pH 8 as background electrolyte with 50 microM CTAB, at 30 degrees C and using a separation voltage of 25 kV. The proposed methodology was then validated for analytical purposes and applied to the analysis of pharmaceutical preparations commercially available. The results obtained with the proposed methodology were in good agreement with those declared by the manufacturers. The simplicity, sample throughput, accuracy, reproducibility and low cost of the proposed method make it suitable for the control of the enantiomeric composition of bupivacaine in pharmaceuticals.     

Optimization of the separation of Vinca alkaloids by nonaqueous capillary electrophoresis

A rapid method for the determination of Vinca alkaloids by nonaqueous capillary electrophoresis with diode array detection has been developed. A group of 11 alkaloids (catharanthine, vinorelbine, anhydrovinblastine, vinflunine, vindoline, 4-O-deacetylvinorelbine, 4-O-deacetylvinflunine, vindesine, vinblastine, 4'-deoxy-20',20'-difluorovinblastine, vincristine) could be readily separated within 10 min. The compounds were separated using a capillary of 38 cm effective length, a running buffer composed of 50 mM ammonium acetate and 0.6 M acetic acid in a methanol-acetonitrile (75:25, v/v) mixture. A constant voltage of 25 kV with a ramp time of 1 min and a 344.7 x 10(3) Pa pressure, applied simultaneously to inlet and outlet buffer vials, were used during sample analysis. Five of these alkaloids were selected for optimization of the separation and for validation studies with respect to specificity, linearity, range, limits of quantification and detection and then accuracy. The feasibility of the assay was demonstrated by analyzing a commercial sample of vinorelbine (Navelbine, ampoule at 10 mg/ml of vinorelbine base). The results were compared with a high-performance liquid chromatography method.     

Recent advances in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis is undergoing rapid development. The scope of the investigations has clearly expanded, covering both basic studies and analytical applications. This review provides a comprehensive overview at the current status of the technique. Special attention is given to theoretical, methodological, and technical challenges, and the achievements of capillary electrophoresis in nonaqueous media are demonstrated with reference to recent applications.     

Efficiency studies in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a relatively new area with several advantages that include enhanced efficiency and improved detection sensitivity. The goal of this study was to investigate the influence of NACE compared to aqueous CE on the separation efficiency of oligosaccharides. The applied voltage and buffer concentration were optimized for the aqueous and nonaqueous buffer media to minimize the band broadening effects of Joule heating and electrophoretic dispersion. At the optimized conditions a 1.5-fold enhancement in efficiency was obtained with the nonaqueous buffer medium.     

Stacking for nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a useful mode in CE for separation and quantification of hydrophobic compounds. However, because of the low conductivity of most of the organic solutions, stacking is not used often in this technique and the sample volume is very limited. As a result of the small sample volume, the detection limits are poor. Furthermore, NACE is affected greatly by the presence of salts in the sample. Here, we show that transient isotachophoresis (t-ITP) can be used easily in this type of electrophoresis to enhance the detection limits and also to reverse the deleterious effects of salts in the sample. Several factors, which affect the stacking in this type of electrophoresis, are described. For example, the presence of salts in the organic solvent, type of sample introduction, and the solvent for the terminating ion were all found to have profound effects on the degree of concentration. Furthermore, the separation time can be shortened by t-ITP.     

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.     

Factors affecting the separation of mandelic acid enantiomers by capillary electrophoresis

Chromatographia - The separation of the optical isomers of mandelic acid by capillary electrophoresis using γ-cyclodextrin as a chiral selector is presented. The mobility of the enantiomers...